Large-scale, economic purification of proteins is an increasingly important concern in the biotechnology industry. Generally, proteins are produced by cell culture using, prokaryotic, e.g., bacterial, or eukaryotic, e.g. mammalian or fungal, cell lines engineered to produce the protein of interest by insertion of a recombinant plasmid comprising the gene for that protein. Since the cell lines used are living organisms, they must be fed with a complex growth medium, comprising sugars, amino acids, and growth factors, sometimes supplied from preparations of animal serum. Separation of the desired recombinant protein from the mixture of compounds fed to the cells and from the by-products generated by the cells themselves to a purity sufficient for use as a human therapeutic poses a formidable challenge.
Multimeric, e.g. homopolymeric and heteropolymeric, proteins represent one of the most complex levels of structural organization in biological molecules. Not only do the constituent polypeptide chains have to fold (into secondary structures and tertiary domains) but they must also form complementary interfaces that allow stable subunit interactions. These interactions are highly specific and can be between identical subunits or between different subunits.
In particular, conventional antibodies are tetrameric proteins composed of two identical light chains and two identical heavy chains. Pure human antibodies of a specific type can be difficult to purify from natural sources in sufficient amounts for many purposes. As a consequence, biotechnology and pharmaceutical companies have turned to recombinant DNA-based methods to prepare antibodies on a large scale. Hundreds of therapeutic monoclonal antibodies (mAbs) are either currently on the market or under development. The production of functional antibodies (including antibody fragments that retain antigen-specificity and often display improved functionality and physico-chemical properties) generally involves the synthesis of the two polypeptides as well as a number of post-translational events, including proteolytic processing of the N-terminal secretion signal sequence; proper folding and assembly of the polypeptides into tetramers; formation of disulfide bonds; and typically includes a specific N-linked glycosylation.
Additionally, cytokines, as pleiotropic regulators that control proliferation, differentiation, and other cellular functions of immune and hematopoietic systems, have potential therapeutic use for a wide range of infectious and autoimmune diseases. Much like antibodies, recombinant expression methods are often used to express recombinant cytokines for subsequent use in research and pharmaceutical applications.
Recombinant synthesis of such proteins has typically relied on cultures of higher eukaryotic cells to produce biologically active material, with cultured mammalian cells being very commonly used. However, mammalian tissue culture-based production systems incur significant added expense and complication relative to microbial fermentation methods. Additionally, products derived from mammalian cell culture may require additional safety testing to ensure freedom from mammalian pathogens (including viruses) that might be present in the cultured cells or animal-derived products used in culture, such as serum.
Prior work has helped to establish the yeast Pichia pastoris as a cost-effective platform for producing functional antibodies that are potentially suitable for research, diagnostic, and therapeutic use. See co-owned U.S. Pat. Nos. 7,935,340; 7,927,863 and 8,268,582, each of which is incorporated by reference herein in its entirety. Methods are also known in the literature for design of P. patoris fermentations for expression of recombinant proteins, with optimization having been described with respect to parameters including cell density, broth volume, substrate feed rate, and the length of each phase of the reaction. See Zhang et al., “Rational Design and Optimization of Fed-Batch and Continuous Fermentations” in Cregg, J. M. Ed., 2007, Pichia Protocols (2nd edition), Methods in Molecular Biology, vol. 389, Humana Press. Totowa, N.J. pgs. 43-63. See also, US 20130045888, entitled MULTI-COPY STRATEGY FOR HIGH-TITER AND HIGH-PURITY PRODUCTION OF MULTI-SUBUNIT PROTEINS SUCH AS ANTIBODIES IN TRANSFORMED MICROBES SUCH AS PICHIA PASTORIS; and US 20120277408, entitled HIGH-PURITY PRODUCTION OF MULTI-SUBUNIT PROTEINS SUCH AS ANTIBODIES IN TRANSFORMED MICROBES SUCH AS PICHIA PASTORIS.
Though recombinant proteins can be produced from cultured cells, undesired side-products may also be produced. For example, the cultured cells may produce the desired protein along with proteins having undesired or aberrant glycosylation. Additionally, cultured cells may produce multi-subunit protein along with free monomers and complexes having incorrect stoichiometry. Purification of the desired multi-subunit protein can increase production cost, and the steps involved in purification may decrease total yield of the desired complex. Moreover, even after purification, undesired side-products may be present in amounts that cause concern. For example, glycosylated side-products may be present in amounts that increase the risk of an immune reaction after administration, and may adversely affect properties such as stability, half-life, and specific activity, whereas aberrant complexes or aggregates may decrease specific activity and may also be potentially immunogenic.